Compositions And Methods For The Detection Of Chemical Warfare Agents

ABSTRACT

Compositions for detection of chemical warfare agents that comprise oximate anion reactive sites and fluorophore cores. Methods for detecting a chemical warfare agents that comprise providing a detector molecule comprising an oximate anion reactive site and a fluorophore core and detecting fluorescence from the detector molecule. Methods for enhancing the reactivity of an oximate nucleophile that comprise introducing an oxime into an aprotic solvent and deprotonating the oxime to form the oximate nucleophile with a base that creates noncoordinating anions.

TECHNICAL FIELD

The present invention, according to specific example embodiments,generally relates to detection of chemical warfare agents using oximefluorophores.

BACKGROUND

It is well established that many organophosphorus compounds are powerfulneurotoxic agents that inhibit acetylcholinesterase (AchE) by theprocess of phosphorylation. A particularly dangerous class oforganophosphorous compounds is the phosphoryl fluoride containingspecies. Two such species are the chemical warfare agents (CWA) sarin(isopropyl methylphosphonofluoridate) and soman (pinacolylmethylphosphonofluoridate), referred to as GB and GD agents,respectively. For obvious safety reasons, CWA may be modeled using achemical warfare agent simulant. Common CWS are diisopropylfluorophosphate (DFP) and diethyl chlorophosphate (DCP).

There has been a significant interest in the decontamination anddetection of CWA over the last five decades, with a large focus onphosphorylfluoride nerve agents e.g., Sarin and Soman. Chemicaldetection of CWA has been a long-term ambition for many researchers,even more so in this day and age due to the continuing global threat ofterrorist activity. One approach that has been studied uses chromogenicdetector reagents, which directly bind to a target nerve agent causing amodulation in the emitted UV-Vis wavelength. However, there arelimitations in the colorimetric systems developed thus far, includinglow sensitivity and slow response times.

One current method for detecting CWA produces a dramatic spectral changecreated in response to the cyclization of a flexible chromophore. See S.W. Zhang & T. Swager, J. Am. Chem. Soc. 125, 3420 (2005). The systemcreates a rigid and highly conjugated fluorophore on the addition ofDFP, causing an “off-on” response in the micromolar concentration range.However, the system utilizes an alcohol as a nucleophile, and hence therate of reaction with DFP, let alone that anticipated with sarin/soman,is quite slow (half-life approaching an hour).

DRAWINGS

A more complete understanding of this disclosure may be acquired byreferring to the following description taken in combination with theaccompanying figures in which:

FIG. 1 shows an example of a reaction mechanism;

FIG. 2 shows the chemical structures of certain fluorophores;

FIG. 3A shows the UV-Vis and fluorescence spectra of certain examplefluorophores;

FIG. 3B shows the UV-Vis and fluorescence spectra of certain examplefluorophores;

FIG. 4 shows two examples of the chemical structure of a coumarinscaffold;

FIG. 5 shows an example of a synthesis scheme for an examplefluorophore;

FIG. 6 shows the UV-Vis spectra of an example fluorophore;

FIG. 7 shows the fluorescence spectra of an example fluorophore; and

FIG. 8 shows a graph of fluorescence intensity versus time for anexample fluorophore.

FIG. 9 shows an example of the chemical structure of an example oximefluorophore with a Lewis-acid attached.

FIG. 10 shows an example of the chemical structure of an example oximefluorophore with a substituted anthracene fluorophore core.

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

While the present disclosure is susceptible to various modifications andalternative forms, specific example embodiments have been shown in thefigures and are herein described in more detail. It should beunderstood, however, that the description of specific exampleembodiments is not intended to limit the invention to the particularforms disclosed, but on the contrary, this disclosure is to cover allmodifications and equivalents as defined by the appended claims.

DESCRIPTION

These fluorophores may be capable of detecting chemical warfare agents(CWA), such as phosphoryl fluoride nerve agents, at low concentrations.Accordingly, the fluorophores of the present disclosure may be used inapplication such as detection of CWA for military and civilianprotection. Such fluorophores also may be used, among other things, insystems and methods for detecting chemical agents. As used herein, theterm “chemical warfare agent” includes chemical warfare simulant.

The fluorophores of the present disclosure generally comprise afluorophore core having an oximate anion as the reactive site. Suchfluorophores may be referred to as “oxime fluorophores.” A fluorophoreis a component of a molecule which causes the molecule to befluorescent. Fluorophore cores suitable for use in the present inventioninclude, but are not limited to, coumarin, fluorescein, substitutedfluoresceins (e.g., esosine), dansyl, rhodamine, anthracene, substitutedanthracenes (e.g., 9,10-diphenyl anthracene), pyrenes, and bodipy. Oneexample of a oxime fluorophore with a substituted anthracene fluorophorecore is shown in FIG. 10.

An oximate anion (RNO⁻) belongs to a class of nucleophiles called “supernucleophiles.” A super nucleophile is a reactive species in which anatom containing an unshared electron pair, typically a nitrogen oroxygen atom, is adjacent to the nucleophilic center. This increases thenucleophilicity of the reactive center, a phenomena commonly known asthe α-effect. Oximate reactive sites can react with the phosphorus(V)center of a CWA. Generally, the oximate anion is formed via thedeprotonation of the oxime (RNOH). Formation of the oximate anion may becarried out by any base strong enough to deprotonate the oxime. In someembodiments, bases that form noncoordinating counterions may be used todeprotonate the oxime. Bases that from noncoordinating counterions may,among other things, enhance the rate of reaction between the oximateanion and the CWA. Examples of suitable bases that form noncoordinatingcounterions include, but are not limited to,1,8-Diazabicyclo[5.4.0]undec-7-ene (“DBU”);1-tert-Butyl-4.4.4.-tris(dimethylamino)-2,2-bis[tris(diethylamino)-phosphoranylidenamino]-2⁵,4⁵-catenadi(phosphazene)(“P₄-t-Bu”);2,8,9-Trimethyl-2,5,8,9-tetraaza-1-phosphabicyclo[3,3,3]undecane(“Verkade base”); and the like.

When oxime fluorophores are deprotonated to form the oximate anion, thehigh energy lone pair orbitals of the oximate anion may quenchfluorescence of the oxime fluorophore via a photoinduced electrontransfer (PET) mechanism. In operation, the oxime fluorophores may thenbe “turned-on” when a chemical agent such as a CWA is added. Uponphosphorylation by a CWA, the energy of these orbitals may bedramatically lowered, thereby reducing the PET quenching effect andturning on the fluorescence. The general reaction mechanism between anoximate and DFP is shown in FIG. 1. In some embodiments, the strength ofthe fluorescence signal may be increased by attaching an amino group tothe fluorophore core. Suitable amino groups include, but are not limitedto, a primary amino group (NH₂), a dimethyl amino group (N(CH₃)₂), and adiethyl amino group (N(CH₂CH₃)₂).

The kinetics of the phosphorylation reaction may be increased byincorporating into the fluorophores a second functional group that has ahigh affinity for fluoride, i.e., a fluoride scavenger moiety. This mayovercome the slower kinetics caused by the strength of thephosphorous-fluoride bond. For example, an average P—Cl bonddissociation energy is 326 KJ/mol, while that for P—F is 490 KJ/mol.Typical fluoride scavenger moieties are Lewis acidic groups such asboronates and pseudo-Lewis acid moieties such as silanes. An example ofan oxime fluorophore with a Lewis-acid attached is shown in FIG. 9. Thesilyl group is excellent in scavenging fluoride, in that this is thecommon procedure for silyl group deprotection. In some embodiments, afluoride scavenger moiety such as silver may be added to a solutioncontaining the oxime fluorophore. By way of explanation, and not oflimitation, the fluoride scavenger moiety may coordinate the P—F bondprior to nucleophilic attack to weaken the bond enough to accomplishrapid detection. A reduction of the half-life to seconds only requiresapproximately a 12 to 17 KJ/mol reduction in activation energy foroximate attack.

Examples of certain oxime fluorophores of the present disclosure areshown in FIG. 2 (compounds 1-2a,2c-4). Such oxime fluorophores may besynthesized using known procedures of synthetic organic chemistry, suchas, for example, the synthetic procedures described in K. J. Wallace, etal., “Colorimetric detection of chemical warfare simulants,” New J.Chem. 29 1469-74 (2005).

In some embodiments, a method for enhancing the reactivity of an oximatenucleophile comprises: introducing the oxime fluorophore into an aproticsolvent and adding a base to deprotonate the oxime fluorophore to formthe oximate, wherein the base forms a noncoordinating counterion.Embodiments of this type may be said to create a “naked” nucleophile,thereby increasing the nucleophilicity and the rate of reaction.Suitable aprotic solvents may be polar or nonpolar. Examples of suitablesolvents include, but are not limited to, DMF, DMSO, acetonitrile, andTHF. Examples of suitable bases include, but are not limited to, DBU,P₄-t-Bu, and Verkade base.

To facilitate a better understanding of the present invention, thefollowing examples of specific embodiments are given. In no way shouldthe following examples be read to limit or define the entire scope ofthe invention.

EXAMPLES

To study certain oxime fluorophores of the present disclosure, a UV-Visabsorbance spectral change needed to be observed. Accordingly, certainexample oxime fluorophores may include a nitro moiety as a UV-Vis‘handle’.

Specific example embodiments of oxime fluorophores may react withsarin/soman chemical warfare simulant in less than about 5 seconds inDMSO, resulting in large emission intensity and wavelength shifts.Fluorescein bisoxime (FIG. 2, compound 2a) and fluorescein monooxime(FIG. 2, compound 2c) both undergo an absorbance shift (a hypsochromicshift) when treated with DFP. See FIG. 1. As a control, a diprotectedspecies (FIG. 2, compound 2b) was synthesized; and for this species, nospectral change was observed. The fluorescence signal of (FIG. 2,compound 2a) is turned off under basic conditions. This may be due tothe lone pair quenching by the super nucleophile, as a consequence ofthe PET mechanism. A fluorescence signal is subsequently ‘turned-on’ bythe addition of DFP. The UV-Vis and fluorescence spectra for compounds2a-2c of FIG. 2 are shown in FIG. 3.

Other specific example embodiments of oxime fluorophores are shown inFIG. 2, compounds 1 and 3. These compounds have a coumarin scaffold (twoexamples are shown in FIG. 4), and many functional groups can beappended to the coumarin scaffold in the four-position. One example of asuitable synthesis for compound 3 of FIG. 2 is shown in FIG. 5.

The specific example oxime fluorophore shown in FIG. 2, compound 1 wasstudied to elucidate some UV-Vis and fluorescence properties. UV-Visstudies were carried out by preparing a solution of compound 1 (FIG. 2)in DMSO (2.5×10⁻⁵ mol dm⁻³). The initial UV-Vis spectra showed a broadband at λ_(max)=409 nm, assigned to the n-π* transition. Upon additionof P₄-t-Bu (^(DMSO)pK_(BH)+=30.25) solution, a bathochromoic shift inwavelength to λ_(max)=443 nm was observed (shown in FIG. 6), typical ofanionic species in solution. On the addition of DFP (6.0 mol dm⁻³ inDMSO) the absorbance intensity is hypsochromically shifted toλ_(max)=409 nm (shown in FIG. 6).

The compound 1 of FIG. 2 is also highly fluorescent, and thefluorescence signal is turned off under basic conditions. This may bedue to PET quenching by the lone pair of the oximate anion. Afluorescence signal is subsequently “turned-on” by the addition of DFP,as shown in FIG. 7. Fluorescence studies (λ_(ex)=410 nm) were carriedout by preparing a 0.5×10⁻⁶ mol dm⁻³ solution in DMSO with a 50 foldexcess of P₄ base, and titrating small aliquots of DFP. FIG. 7 shows thefluorescence signal of compound 1 with the P₄-t-Bu base alone shows tobe a weak fluorescence signal. The fluorescence signal increases withthe addition of DFP.

Stop-flow kinetics experiments were carried out by watching the “turnon” of the fluorescence signal upon the addition of DFP. A 2.5×10⁻⁵ moldm⁻³ of compound 1 of FIG. 2 was prepared in DMSO with the P₄-t-Bu base.A 1.25×10 mol dm⁻³ of DFP was prepared in DMSO. One milliliter of eachsolution was transferred to a separate syringe and placed in thestop-flow apparatus. Equal volumes of the solutions were mixed togetherand the reaction was monitored for 1 second. The fluorescence intensityincreased upon mixing, in good agreement with the fluorescence studiesdescribed above. By monitoring the fluorescence intensity at varioustimes one can calculate the rate of the reaction by plotting1n(A_(o)/(A_(o)−P)) versus time (FIG. 8 inset). Where A_(o) is the finalfluorescence intensity and P is the fluorescence intensity at each timeinterval measured. The rate constant k (slope) was calculated to be 1410s⁻¹. Therefore the half-life (t_(1/2))=1n(2)/k, is calculated to beapproximately 50 ms.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in itsrespective testing measurements.

Therefore, the present invention is well adapted to attain the ends andadvantages mentioned as well as those that are inherent therein. Whilenumerous changes may be made by those skilled in the art, such changesare encompassed within the spirit of this invention as illustrated, inpart, by the appended claims.

1. A composition for detection of a chemical warfare agent comprising anoximate anion reactive site and a fluorophore core.
 2. The compositionof claim 1, wherein the fluorophore core comprises at least onefluorophore chosen from coumarin, fluorescein, a substitutedfluorescein, dansyl, rhodamine, anthracene, a substituted anthracene, apyrene, and bodipy.
 3. The composition of claim 1, further comprising afluoride scavenger moiety.
 4. The composition of claim 1, furthercomprising at least one fluoride scavenger moiety chosen from a Lewisacid group, a pseudo-Lewis acid moiety, a boronate, and a silane.
 5. Thecomposition of claim 1, further comprising a primary amino, a dimethylamino, or a diethylamino group.
 6. A composition for detection of achemical warfare agent comprising a compound represented by thefollowing formula:

wherein X is a structure represented by


7. A composition for detection of a chemical warfare agent comprising acompound represented by the following formula:

wherein at least one of X or X′ is H.
 8. A composition for detection ofa chemical warfare agent comprising a compound represented by thefollowing formula:


9. A composition for detection of a chemical warfare agent comprising acompound represented by the following formula:

wherein X and Y are independently H, an alkyl, a phenyl, OH, OR, NH₂,NR₂, NO₂, CN, CO₂H, an ester, and an amide, and wherein R is ahydrocarbon.
 10. A method for detecting a chemical warfare agentcomprising: providing a detector molecule comprising an oximate anionreactive site and a fluorophore core; and detecting fluorescence fromthe detector molecule.
 11. The method of claim 10, wherein thefluorophore core comprises at least one fluorophore chosen fromcoumarin, fluorescein, a substituted fluorescein, dansyl, rhodamine,anthracene, a substituted anthracene, a pyrene, and bodipy.
 12. Themethod of claim 10, wherein the detector molecule further comprises atleast one fluoride scavenger moiety chosen from a Lewis acid group, apseudo-Lewis acid moiety, a boronate, and a silane.
 13. The method ofclaim 10, further comprising providing a fluoride scavenger in solution.14. The method of claim 10, further comprising providing a fluoridescavenger in solution, wherein the fluoride scavenger is silver.
 15. Themethod of claim 10, further comprising a primary amino, a dimethylamino, or a diethylamino group.
 16. A method for enhancing thereactivity of an oximate nucleophile comprising: introducing an oximeinto an aprotic solvent; and deprotonating the oxime to form the oximatenucleophile with a base that creates noncoordinating counterions. 17.The method of claim 16, wherein the base that creates noncoordinatingcounterions comprises at least one base chosen from1,8-Diazabicyclo[5.4.0]undec-7-ene;1-tert-Butyl-4.4.4.-tris(dimethylamino)-2,2-bis[tris(diethylamino)-phosphoranylidenamino]-2⁵,4⁵-catenadi(phosphazene);and 2,8,9-Trimethyl-2,5,8,9-tetraaza-1-phosphabicyclo[3,3,3]undecane.